Process for producing soybean protein hydrolysate

ABSTRACT

An object of the present invention is to provide a process for producing a sterilized enzymatic decomposition soybean protein with facilitating separation of a precipitate after decomposition of soybean protein with an enzyme, and with improving a yield of the enzymatic decomposition product, thereby minimizing formation of the dregs upon using in drinks and so on. Said process comprises adding a proteolytic enzyme to a soybean protein solution to effect hydrolysis, heating and cooling the hydrolyzation mixture, separating and removing insolubles from the mixture and heat-sterilizing the resultant supernatant.

FIELD OF THE INVENTION

The present invention relates to a process for producing a soybeanprotein hydrolysate. More specifically, it relates to a process forproducing a soybean protein hydrolyzed with an enzyme in a high yieldwith minimizing formation of dregs upon dissolution.

BACKGROUND ART

Products obtained by hydrolyzing proteins with a proteolytic enzyme havebetter absorbability upon digestion than that of proteins withoutproteolysis, and are utilized in various fields such as health food andthe like. In particular, it is expected to use them in sports drinks,drinks for nutrition, and the like.

Up to now, JP 61-254153 A, JP 1-269499 A, JP 2-23885 A, 4-190797 A, JP8-322471 A, JP 10-271958 A, etc. disclose processes for producingenzymatic decomposition products obtained by hydrolyzing animal andvegetable proteins with enzymes. In general, after hydrolysis ofproteins with an enzyme, heat treatment is carried out in these knownprocesses so as to inactivate the enzyme, sterilizing the products, andso on. In particular, anaerobic thermophilic bacteria often causeproblems in these kinds of products and, normally, thorough heatsterilization is required.

In addition, although a water-soluble fraction and a water-insolublefraction are separated after hydrolysis, conventional drinks containingprotein hydrolysates are liable to form a small amount of a precipitate(dregs) during storage and this is a problem. In general, improvement ofquality causes decrease in a yield, whereas increase in a yield isliable to form more dregs during storage. This is also a problem.

OBJECTS OF THE INVENTION

One object of the present invention is to provide a process forproducing a soybean protein hydrolyzed with an enzyme in a high yieldwith minimizing formation of dregs upon dissolution.

SUMMARY OF THE INVENTION

The present inventors have studied intensively to solve the aboveproblems. As a result, it has been found that the above problems can besolved by two step heat treatment, wherein, after hydrolysis of aprotein with an enzyme, the hydrolyzation mixture is subjected to a stepfor heating lightly, followed by cooling to separate insolubles beforesubjecting the mixture to heat sterilization. Thus, the presentinvention has been completed.

That is, the present invention is a process for producing a soybeanprotein hydrolysate which comprises hydrolyzing a soybean proteinsolution with a proteolytic enzyme, heating (a) and cooling thehydrolyzation mixture, separating and removing insolubles from themixture to obtain a supernatant and heat-sterilizing (b) thesupernatant.

In comparison with heat treatment generally employed for inactivating anenzyme or sterilization, the above heating (a) is preferably carried outunder such mild conditions that heating time is 10^(5.25−(0.05×T))minutes (wherein T is heating temperature (°C.)) or shorter, becauseheat sterilization (b) is carried out afterward. Inactivation of theenzyme and sterilization can be effected by heat sterilization (b) ofthe supernatant after separating and removing insolubles.

Preferably, heating (a) is carried out until the temperature rises to75° C. or higher, and cooling is carried out until the temperature dropsto 60° C. or lower. Preferably, insolubles are separated and removed ata pH of the soybean protein solution of 4.0 to 6.2, or the soybeanprotein solution contains an alkaline earth metal compound or a proteinflocculating agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As a raw material for preparing the soybean protein solution of thepresent invention, which is derived from soybeans and is availableinexpensively, there can be used soybean milk, concentrated soybeanprotein, isolated soybean protein, defatted-soybeans, soybean wheyprotein and the like. Among them, soybean milk or isolated soybeanprotein is preferred. When concentrated soybean protein ordefatted-soybeans is used, separation of “okara (insoluble residue)”after enzymatic decomposition tends to be difficult. And, it takes muchtime to collect whey protein, and whey protein has an inferior flavor.As an alkali to be used for preparing the soybean protein solution, oradjusting pH of the hydrolyzation mixture, sodium hydroxide can be used.Potassium hydroxide can also be used in view of nutrition. As an acid,preferably, an organic acid such as citric acid is used in view of aflavor.

The concentration of the soybean protein solution to be subjected to theenzymatic treatment is 1 to 30% by weight, preferably 5 to 15% byweight, more preferably 8 to 12% by weight. Even if the concentration islow, it will not be an obstacle to the process itself. However, theproductivity is lowered, which causes increase in the production cost ofa soybean protein hydrolysate. When the concentration of the soybeanprotein solution is too high, a large amount of an enzyme is requiredfor decomposing the protein sufficiently. This may be caused bypolymerization of protein hydrolysates once formed by hydrolysis oneanother, and is undesirable.

As the proteolytic enzyme to be used in the present invention(protease), an exoprotease or endoprotease can be used alone or incombination thereof. The enzyme may be that derived from animals,vegetables or microorganisms. Specifically, serine proteases (trypsin,chymotrypsin, etc. derived from animals; subtilisin, carboxypeptidase,etc. derived from microorganisms; etc.), thiol proteases (papain ficin,bromelain, etc. derived from vegetables) and carboxy proteases (pepsinderived from animals) can be used. Further, the specific examplesthereof include Protin FN (trade name of protease manufactured byDaiwakasei K. K.) derived from Aspergillus oryzae, Actinase (trade nameof protease manufactured by Kaken Seiyaku K. K.) derived fromStreptomyces griseus, Alkalase (trade name of protease manufactured byNovo) derived from Bacillus licheniformis, Protin A (trade name ofprotease manufactued by Daiwakasei K. K.) derived from Bacillussubtilis, and the like. In addition, examples of enzyme preparationscontaining endoproteases include Protease S manufactured by AmanoSeiyaku K. K., Protin AC-10 manufactured by Daiwakasei K. K. and thelike. Examples of proteolytic enzymes containing exo- and endoproteasesinclude Protease M manufacuted by Amano Seiyaku K. K.

Conditions for hydrolysis of the present invention vary to some extentaccording to a particular kind of the proteolytic enzyme to be used.However, in general, it is preferred to use the enzyme in an amountsufficient for hydrolyzing soybean protein in a pH range at atemperature effective for the enzyme activity. When pH is 5 to 10,preferably 6 to 9, formation of a salt by neutralization can be reducedand this is desired in view of using the hydrolysate for asalt-restriction diet (e.g., alimental infusion, etc.).

The degree of hydrolysis is about 20 to 98%, more preferably about 50 to90% in terms of a soybean protein decomposition rate expressed by asolubilization degree of a protein component in 15% trichloroaceticacid. Although time for acting a proteolytic enzyme varies dependingupon the activity of a particular proteolytic enzyme to be used and itsamount, normally, it may be about 5 minutes to 24 hours, preferablyabout 30 minutes to 9 hours. When the enzymatic decomposition time istoo long, putrefaction is liable to take place.

The hydrolyzed soybean protein solution is subjected to heating (a) andcooling prior to the step for separating and removing insolublestherefrom. This heating (a) is effected lightly in comparison with thatfor heat sterilization. When this heating is effected excessively sothat the heating time is in excess of 10^(5.25−(0.05×T)) minutes orshorter, a material causing dregs upon dissolution of the decomposedproduct is formed, presumably, due to formation of a fraction elutedfrom a precipitated fraction of the soybean protein hydrolysate formedby hydrolysis. This is undesirable. On the contrary, when this heatingis not effected, flocculation capability of insolubles is poor, whichresults in a difficulty in separation between a supernatant andinsolubles with a practical continuous separation means. Flocculationcapability can be readily judged by, for example, collecting a 10 ccsample of an enzyme hydrolyzation mixture at 25° C., which is resultedfrom enzymatic decomposition of a soybean protein solution, followed byheat treatment, in a graduated centrifuge tube, centrifuging at 1,500 Gfor 20 minutes to precipitate a sludge (precipitate) and then comparingthe volume of the sludge to that of a sample obtained according to thesame manner except that the heat treatment is not effected.Specifically, it is preferred to effect this heating in such a degreethat the volume of the former is ⅔ or less of the latter. The requiredheating time can be readily determined within the range of 75 to 160°C., preferably 80 to 140° C. Shorter heating time can be employed as aheating temperature is higher. Normally, the heating time (minutes) of10^(5.25−(0.05×T)) or longer is sufficient. For example, it issufficient to raise a temperature up to about 110° C., followed bymaintaining this temperature for about 0.01 minute and then cooling.

It is suitable to effect the cooling of the next step so that thetemperature is dropped to 60° C. or lower, preferably 30° C. or lower,more preferably 15° C. or lower. When this cooling is omitted, it makesthe separation of insolubles difficult. Then, in case of centrifugation,a high centrifugal force or longer holding time is required forseparation of insolubles. Namely, when heating and cooling were notcarried out prior to separation, longer holding time such as for 20minutes at 1,500 g was required for separation of insolubles bycentrifugation. However, according to the present invention, insolublescan be separated by centrifugation within shorter holding time such asfor several seconds to 5 minutes at 1,500 g. Therefore, according to thepresent invention, continuous centrifugation can be employed, whereascontinuous centrifugation is hardly employed in a conventional process.In addition, as to the degree of insolubilization, a larger amount ofdregs tend to form as the difference between the heating temperature andthat of a cooling medium becomes larger. Then, as the coolingconditions, it is preferred that difference between the heatingtemperature and that of a cooling medium is larger.

Further, although cooling can be carried out by allowing to stand(naturally cooling), artificial cooling with a cooling medium ispreferred because cooling can be carried out quickly to insolubilizecomponents of dregs quickly, thereby facilitating prevention offormation of dregs upon dissolution of the product.

Separation of insolubles can be carried out by a filtration means suchas a filter press, membrane filter, or the like. However, normally,centrifugation is employed and, in particular, a continuous centrifugalseparator, a liquid cyclone, etc. can be used.

Normally, pH of the hydrolyzation mixture is within the range of 3 to 8.In order to accelerate or improve separation/flocculation capability ofthe above insolubles, it is suitable that the pH is preferably 4 to 6.2,more preferably 4.5 to 5.5 because insolubles containing undecomposedmaterials tend to flocculate at about isoelectric point of soybeanprotein. Alternatively, separation/above flocculation capability canalso be accelerate or improved by addition of an alkaline earth metalcompound such as a salt, for example, chloride or sulfate, or ahydroxide of calcium, magnesium, etc. or a flocculating agent such assodium polyacrylate, alginic acid, chitin, chitosan, etc. to thehydrolyzation mixture.

After separation and removal of insolubles, heat sterilization (b) iscarried out. This treatment can be carried out according to a knownmethod. However, when the above heating (a) is effective for heatsterilization and inactivation of the enzyme though it is weak,conditions of this heating can be milder by taking into consideration ofthe effect of the heating (a). Suitably, this heating is carried out insuch a degree that the enzyme remained in the soybean proteinhydrolysate is substantially inactivated and a remaining viable count is10 or less. Normally, it is preferred to carry out heating in excess ofthe above-described heating time, i.e., for longer than10^(5.25−(0.05×T)) minutes (wherein T is heating temperature (° C.)).

The pH of the hydrolyzation mixture to be subjected to this heatsterilization (b) is preferably determined by a particular use of theend product and, normally, it is within the range of pH 3 to 8. When thehydrolysate of the present is used for neutral drinks, preferably, theend product is within pH 6 to 7. When the hydrolysate is used for acidicdrinks, pH 3.5 to 4.5 is suitable. The degree of sterilization varies tosome extent according to this pH. In case of weak acidic to neutral,sterilization of thermophilic anearobes such as Clostridium, etc. isrequired and, preferably, the heating is carried out in excess of, i.e.,for longer than 10^(6.25−(0.05×T)) minutes. On the other hand, when thefinal pH is about 4.5 or lower, the heating time of 10^(6.25−(0.05×T))minutes or shorter is sufficient because growth of almost all pathogenicbacteria, putrefactive bacteria and sporangia are hardly taken place.

The product resulted from the heat sterilization (b) can be stored as itis, or after concentration, by sealing in a container. Alternatively,the product can be dried and pulverized or atomized for storage.

EXAMPLES

The embodiments of the present invention are illustrated by thefollowing Examples.

Example 1

An aqueous 0.9% solution (pH 7.0) of isolated soybean protein (“New FujiPro-R” manufactured by Fuji Oil, Co., Ltd.)(30 kg) was prepared andsubjected to an enzymatic reaction with a proteolytic enzyme (“ProteaseS” manufactured by Amano Seiyaku K. K.) (1.2 kg) to hydrolyze theprotein at 60° C. for 5 hours (15% TCA solubilization degree: 85%).Then, the hydrolyzation mixture was adjusted to pH 5.5 by addition ofcitric acid. Steam at 8 kg/cm² was blown into the mixture to raise itstemperature to 95° C. and the mixture was held at this temperature for 1minute (heating (a)). The mixture was cooled to 12° C. with a heatexchanger plate through which cooling water was passing, followed bycentrifugation with a high-speed continuous centrifugal separator (SB-7manufactured by WESTFALLIA SEPARATOR) by adjusting a feed rate to 100L/hour to separate and remove a precipitate fraction formed. Theprecipitate fraction was sufficiently firm to retain it for 20 minutesuntil it was discharged. The resultant supernatant (yield of solids:72.4%) was adjusted to pH 6.5 and sterilized at 150° C. for 1 minute(heat-sterilization (b)). Immediately after sterilization, thesupernatant was spray-dried to obtain a dried powder. The resultantdried powder was dissolved in water at a concentration of 5%. When thissolution was stored at 5° C. for 24 hours, dregs were not observed atall.

The above hydrolyzation mixture (10 cc) was collected in a gradedcentrifuge tube before heating (a) and adjusted its temperature to 25°C. and centrifuged at 1,500 G for 20 minutes. The volume of a sludge was32% of that of the hydrolyzation mixture. By heating (a), the volume ofa sludge was condensed to 10% of that of the hydrolyzation mixture.

Comparative Example 1 The First Heating Step was Omitted

According to the same manner as that described in Example 1, a driedpowder was obtained except that the soybean protein solution adjusted topH 5.5 was not subjected to the heat treatment, but was directly cooledto 12° C. with a heat exchanger plate, followed by centrifugation. Inthis case, the precipitate fraction formed in the centrifugal separatorwas insufficiently firm. Then, the fraction could be retained only for 5minutes until it was discharged. The resultant dried powder wasdissolved in water at a concentration of 5% and the solution was storedat 5° C. for 24 hours. Although dregs were not formed at all, the yieldof solids of the supernatant was only 53.2%.

Comparative Examples 2 and 3 Heat Sterilization was Effected as theFirst Heating Step

According to the same manner as that described in Example 1, a driedpowder was obtained except that the hydrolyzation mixture was raised to95° C. and maintained at that temperature for 20 minutes, or raised to80° C. and maintained at that temperature for 60 minutes instead ofraising to 95° C. and maintaining at that temperature for 1 minute, andthat heat sterilization was omitted. The volume of a sludge determinedby adjusting the temperature to 25° C. and centrifuging at 1,500 G for20 minutes was almost the same as that of Example 1. However, when theresultant dried powder was dissolved in water at a concentration of 5%and the solution was stored at 5° C. for 24 hours, dregs were clearlyrecognized with the naked eye in both cases.

Example 2 and Comparative Example 4 Cooling was Slow Cooling or Omitted

According to the same manner as that described in Example 1, a driedpowder was obtained except that the hydrolyzation mixture was allowed tocool for 4 hours to room temperature and then centrifuged (Example 2),or was centrifuged directly without cooling (Comparative Example 4)instead of subjecting to heat treatment at 95° C. for 1 minute, coolingto 12° C. and then centrifugation.

The dried powder was dissolved in water at a concentration of 5% andstored at 5° C. for 24 hours. As a result, dregs were slightly formed inExample 1, whereas dregs were clearly formed in Comparative Example 4.

Example 3 and Comparative Examples 5 and 6

According to the same manner as that described in Example 1, a driedpowder was prepared except that the hydrolyzation mixture was adjustedto pH 4.5 by addition of citric acid, the heating (a) was carried out byholding at 100° C. for 6 seconds, that the mixture was cooled to 15° C.,that the centrifugation was carried out a continuous centrifugalseparator (MD-10 manufactured by IshikawajimaHarima Heavy IndustriesCo., Ltd.) by adjusting a feed rate to 30 L/hour, and that the heatsterilization (b) was carried out at 125° C. for 10 seconds (Example 3).When the dried powder was dissolved in water at a concentration of 5%and stored at 5° C. for 24 hours, dregs were not formed at all.

The volume of a sludge determined before heating (a) by raising thetemperature to 25° C. and centrifuging at 1,500 G for 20 minutes was 30%based on the volume of the hydrolyzation mixture, whereas the volume ofa sludge determined after heating (b) was 10% based on the volume of thehydrolyzation mixture. The yield of the supernatant by continuouscentrifugation was 65%.

The soybean protein was treated according to the same manner as thatdescribed in Example 3 except that the heating (a) was omitted(Comparative Example 5). However, separation with the continuouscentrifugal separator was bad. In addition, according to the same manneras that described in Example 3, a dried powder was produced except thatthe heating (a) was carried out by holding at 104° C. for 5 minutes(Comparative Example 6). When the dried powder was dissolved in water ata concentration of 5% and stored at 5° C. for 24 hours, dregs wereclearly formed.

Example 4 and Comparative Example 7

An aqueous 9% soybean protein solution (pH 7.0) was prepared by usingthe same isolated soybean protein as that in Example 1 (30 kg) andsubjected to an enzymatic reaction with a proteolytic enzyme (“ProteaseM” manufactured by Amano Seiyaku K. K.) (E/S ratio=2%) in a continuousenzymatic reaction vessel for 2 hours. After addition of CaSO₄ in anamount 0.5% by weight based on the weight of the substrate, steam wasblown into the hydrolyzation mixture so that the temperature was raisedto 130° C. and heating (a) was stopped. The mixture was cooled to 15° C.with a heat exchanger plate through which cooling water was passing,followed by treatment with a continuous separator, a liquid cyclone(NHC-10 manufactured by Nippon Kagaku Kikai Seizo) by adjusting a feedrate to 400 L/hour to separate and remove insoluble components. Theresultant supernatant was adjusted to pH 6.5 and sterilized at 150° C.for 1 minute. Immediately after sterilization, the supernatant wasspray-dried to obtain a dried powder. The resultant dried powder wasdissolved in water at a concentration of 5%. When this solution wasstored at 5° C. for 24 hours, dregs were not formed at all.

As Comparative Example 7, the soybean protein was treated according tothe same manner as that of Example 4 except that heating to 130° C. byblowing steam into the hydrolyzation mixture was omitted and the mixturewas directly cooled to 15° C. However, the insoluble components couldnot be separated by using the liquid cyclone.

Effect of the Invention

According to the present invention, a precipitate formed after enzymaticdecomposition of soybean protein is readily separated, thereby improvinga yield. And, when the resultant enzymatic decomposition product is usedfor drinks, formation of dregs can be minimized.

What is claimed is:
 1. A process for producing a soybean proteinhydrolysate, which comprises: (i) hydrolyzing an aqueous soybean proteinsolution with a proteolytic enzyme to form a hydrolyzed mixture, (ii)subjecting the hydrolyzed mixture to heating at 75 to 160° C. for10^(5.25−(0.05×T)) minutes or less wherein T is the heating temperature(° C.) to form a heated hydrolyzed mixture, (iii) cooling the heatedhydrolyzed mixture to 60° C. or less, (iv) separating and removinginsolubles from the cooled mixture at a pH of 4.0-6.2 to obtain asupernatant, and (v) subjecting the supernatant to heat-sterilization,to obtain the soybean protein hydrolysate.
 2. The process according toclaim 1, wherein the hydrolyzing step is carried out so that the degreeof hydrolysis is 20 to 98% in terms of a soybean protein decompositionrate expressed by a solubilization degree of a protein component in 15%trichloroacetic acid.
 3. The process according to claim 1, wherein theinsolubles are separated and removed by addition of an alkaline earthmetal compound or a protein flocculating agent.